Before implanting into the uterine wall, the mammalian conceptus specifies the cell types that are the founders of trophoblast, extra-embryonic endoderm, and fetus. The first morphologically distinct cell type of the trophoblast lineage is the trophectoderm, which becomes discernible at the morula stage and gives rise to the placental trophoblast. The first morphologically distinct cell type of the extra-embryonic endoderm is the primitive endoderm, which at the late blastocyst stage becomes visible as a cell layer on the mural surface of the Inner Cell Mass (ICM) and gives rise to the yolk sac endoderm with its visceral and parietal components. Finally, the first morphologically distinct cell type of the fetal lineage is the epiblast, which constitutes the remainder of the late ICM and gives rise to amnion, extra-embryonic mesoderm, and embryo proper (Nagy et al., Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed, Cold Spring, N.Y.: Cold Spring Harbor Laboratory Press p. 764 (2002)).
Cultured cell lines that maintain or acquire pre- or peri-implantation embryo cell type are the well-known mouse embryonic stem (ES) cells (Evans et al., Nature 292:154-156 (1981) and Martin G., Proc Natl Acad Sci USA 78:7634-7638 (1981)), which closely resemble the nascent epiblast (Nichols et al., Development [Epub ahead of print] Aug. 26 (2009)).
The situation has been less clear regarding cell lines representing extra-embryonic endoderm lineages. Cell lines with trophoblastic (and perhaps extra-embryonic-endodermal) differentiation potential (Buehr et al., Biol Reprod 68:222-229 (2003); Fandrich et al., Nat Med 8:171-178 (2002); and Epple-Farmer et al., Cell Transplant April 29.pii:CT-1966 [Epub ahead of print]) have been derived from rat blastocysts. Furthermore, extra-embryonic endoderm stem cell lines called “XEN cells” (“XEN” for extra-embryonic endoderm) have been isolated from mouse blastocysts (Kunath et al., Development 132:1649-1661 (2005)). These XEN cells can efficiently contribute to parietal endoderm in vivo, but they did not efficiently integrate into the visceral endoderm (i.e., the committed extra-embryonic endoderm precursor). XEN cells do not express the transcription factor Oct4 (Kunath et al. (2005)) that is found in all cells of the early ICM (Ovitt et al., Mol Hum Reprod 4:1021-1031 (1998)).
One analysis of mouse blastocysts has raised the possibility that the committed extra-embryonic endoderm precursor exists already in the early ICM (Chazaud et al., Dev Cell 10:615-624 (2006) and Kurimoto et al., Nucleic Acids Res 34:e42 (2006)).
Debeb et al., PLoS One 4: e7216 (2009) has shown that, from rat blastocysts, cell lines with extra-embryonic endoderm identity can be derived that are distinguished from XEN cells by a less mature marker spectrum (including Oct4) and a better ability to form visceral endoderm (in addition to parietal) in vitro and in vivo. These cells appear to represent the first committed step of the extra-embryonic endoderm lineage, and were, therefore, named XEN-F cells (“F” for precursor).
Debeb generated the rat cell lines that express extra-embryonic endodermal precursor markers by explanting the blastocysts onto mitotically-inactivated primary embryo fibroblasts (generally designated in the industry as a “feeder layer”).